Effect of pectin enhancement on plasma quercetin and fecal flora in rutin-supplemented mice.

Few reports have considered the effects of dietary fiber on plasma quercetin and the intestinal flora. We investigated the effects of pectin on the plasma and fecal flora of mice fed a diet supplemented with the quercetin glycoside rutin. Male mice were randomly divided into 2 groups, which were fed a pectin-rutin (PR) or cellulose-rutin (CR) diet for 14 d. Plasma quercetin and isorhamnetin metabolites were measured by high-performance liquid chromatography. Feces were immediately processed with bacteriological procedures. The fecal flora was investigated. Plasma quercetin and isorhamnetin concentrations were significantly higher in the PR diet group, as was the plasma isorhamnetin/quercetin ratio. The composition of the intestinal flora differed between the 2 dietary groups. The total number of fecal bacteria was significantly larger in the PR group, in which most types of bacteria were more abundant, with the exceptions of bifidobacteria, fusiform-shaped bacteria, and staphylococci. The lower gut seemed to be the major absorption site for rutin. Pectin might thus enhance the bioavailability of quercetin from rutin by altering the metabolic activity of the intestinal flora and/or gut physiological function.

Baicalein, a flavonoid extracted from a methanolic extract of Oroxylum indicum inhibits proliferation of a cancer cell line in vitro via induction of apoptosis.

A methanolic extract of the fruits of Oroxylum indicum, which is widely used in traditional Chinese herbal medicine for its anti-inflammatory, anti-pyretic and anti-hypersensitivity effects, inhibited in vitro proliferation of HL-60 cells. The flavonoid baicalein was found as an active component in the extract. Analysis of freeze-dried fruits of the plant indicated that this component comprised about 4% of the material by dry weight. In this study, we investigated the in vitro effects of baicalein on the viability and induction of apoptosis in the HL-60 cell line. The cell viability after treating with baicalein for 24 h was quantified by counting viable cells using trypan blue staining. The results showed that baicalein caused a 50% inhibition of HL-60 cells at concentrations of 25-30 microM. The inhibition of proliferation of HL-60 cells due to 36-48 h exposure to 10 or 20 microM baicalein was associated with the accumulation of cells at S or G2M phases. However, proliferation inhibition at a higher dose may be associated with induction by apoptosis, as evidenced by the typical nuclear fragmentation using DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The results indicate that baicalein has anti-tumor effects on human cancer cells, and Oroxylum indicum extract could be used in supplementary cancer therapy.

Inhibitory effect of a quercetin metabolite, quercetin 3-O-beta-D-glucuronide, on lipid peroxidation in liposomal membranes.

To study the antioxidant activity of quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after intake of quercetin-rich food, the inhibitory effect of Q3GA on lipid peroxidation was estimated using phosphatidylcholine large unilamellar vesicles (PC LUV) as a biomembrane model. Iron ion, an aqueous peroxyl radical generator, a peroxynitrite generator, or lipoxygenase was used as the inducer of lipid peroxidation. In all cases, Q3GA inhibited lipid peroxidation significantly, although its inhibitory effect was lower than that of quercetin aglycon. The ultrafiltration of PC LUV containing Q3GA revealed that Q3GA has low but significant affinity with the membranes of phospholipid bilayers. It is therefore likely that Q3GA acts as an efficient antioxidant in membranous lipid peroxidation through its localization in the phospholipid bilayer. This conjugated quercetin metabolite seems to retain the ability to protect cellular and subcellular membranes from peroxidative attack by reactive oxygen species and peroxidative enzymes.

Structural analysis of a novel antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines.

Six compounds were isolated from fresh rhizomes of fingerroot (Boesenbergia pandurata Schult.) as strong antimutagens toward 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in Salmonella typhimurium TA98. These compounds were 2',4',6'-trihydroxychalcone (pinocembrin chalcone; 1), 2',4'-dihydroxy-6'-methoxychalcone (cardamonin; 2), 5,7-dihydroxyflavanone (pinocembrin; 3), 5-hydroxy-7-methoxyflavanone (pinostrobin; 4), (2,4,6-trihydroxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (5), and (2,6-dihydroxy-4-methoxyphenyl)-[3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl]methanone (panduratin A; 6). Compound 5 was a novel compound (tentatively termed 4-hydroxypanduratin A), and 1 was not previously reported in this plant, whereas 2-4 and 6 were known compounds. The antimutagenic IC(50) values of compounds 1-6 were 5.2 +/- 0.4, 5.9 +/- 0.7, 6.9 +/- 0.8, 5.3 +/- 1.0, 12.7 +/- 0.7, and 12.1 +/- 0.8 microM in the preincubation mixture, respectively. They also similarly inhibited the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). All of them strongly inhibited the N-hydroxylation of Trp-P-2. Thus, the antimutagenic effect of compounds 1-6 was mainly due to the inhibition of the first step of enzymatic activation of heterocyclic amines.

Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin.

The potential beneficial effect of dietary quercetin (3,3',4',5,7-pentahydroxyflavone) has attracted much attention in relation to the prevention of cardiovascular disease. It is generally recognized that dietary quercetin is subject to metabolic conversion resulting in conjugated forms during absorption and circulation. However, no quercetin conjugates have yet been identified from biological fluids or tissues. In the present study, we isolated and characterized two quercetin conjugates from the plasma of quercetin-administered rats. The blood plasma was collected from 26 rats 30 min after oral administration of quercetin (250 mg/kg body weight), concentrated, dissolved in 2% acetic acid aqueous solution (pH 2.65), and extracted with ethyl acetate. Two compounds (P2, P3) were obtained from the extract by repeated reversed-phase HPLC. On the other hand, two quercetin glucuronides were synthesized chemically and identified as quercetin 3-O-beta-D-glucuronide (Q3GA) and quercetin 4'-O-beta-D-glucuronide (Q4'GA), as determined from FABMS, 1H- and 13C-NMR, and HMBC data. The retention times of P2 and P3 in the HPLC chromatogram corresponded to those of Q3GA and Q4'GA, respectively. FABMS data demonstrated that P2 and P3 are quercetin monoglucuronides. 1H-NMR data for P2 were completely in agreement with those for Q3GA. P2 was therefore identified as Q3GA. This is, to our knowledge, the first evidence that Q3GA accumulates in vivo after oral administration of quercetin. Q3GA is likely to act as an effective antioxidant in blood plasma low-density lipoprotein, because this conjugated metabolite was found to possess a substantial antioxidant effect on copper ion-induced oxidation of human plasma low-density lipoprotein as well as 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.

Protection by quercetin and quercetin 3-O-beta-D-glucuronide of peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein.

Effect of quercetin and its conjugated metabolite quercetin 3-O-beta-D-glucuronide (Q3GA), on peroxynitrite-induced consumption of lipophilic antioxidants in human plasma low-density lipoprotein (LDL) was measured to estimate the role of dietary flavonoids in the defense system against oxidative modification of LDL based on the reaction of nitric oxide and superoxide anion. Synthesized peroxynitrite-induced consumption of endogenous lycopene beta-carotene and alpha-tocopherol was effectively suppressed by adding quercetin aglycone into LDL solution. Q3GA also inhibited the consumption of these antioxidants effectively. These results indicate that dietary quercetin is capable of inhibiting peroxynitrite-induced oxidative modification of LDL in association with lipophilic antioxidants present within this lipoprotein particle.

Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells.

We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly. Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.

Inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation in human low-density lipoprotein.

To determine the antioxidant activity of dietary quercetin (3,3',4', 5,7-pentahydroxyflavone) in the blood circulation, we measured the inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation of human low-density lipoprotein (LDL). Conjugated quercetin metabolites were prepared from the plasma of rat 1 h after oral administration of quercetin aglycone (40 micromol/rat). The rate of cholesteryl ester hydroperoxide (CE-OOH) accumulation and the rate of alpha-tocopherol consumption in mixtures of LDL solution (0.4 mg/ml) with equal volumes of this preparation were slower than the rates in mixtures of LDL with preparations from control rats. The concentrations of CE-OOH after 2 h oxidation in the mixtures of LDL with preparations of conjugated quercetin metabolites were significantly lower than those in the control preparation. It is therefore confirmed that conjugated quercetin metabolites have an inhibitory effect on copper ion-induced lipid peroxidation in human LDL. Quercetin 7-O-beta-glucopyranoside (Q7G) and rhamnetin (3,3',4', 5-tetrahydroxy-7-methoxyflavone) exerted strong inhibition and their effect continued even after complete consumption, similarly to quercetin aglycone. The effect of quercetin 3-O-beta-glucopyranoside (Q3G) did not continue after its complete consumption, indicating that the antioxidant mechanism of quercetin conjugates lacking a free hydroxyl group at the 3-position is different from that of the other quercetin conjugates. The result that 4'-O-beta-glucopyranoside (Q4'G) and isorhamnetin (3,4',5, 7-tetrahydroxy-3'-methoxyflavone) showed little inhibition implies that introduction of a conjugate group to the position of the dihydroxyl group in the B ring markedly decreases the inhibitory effect. The results of azo radical-induced lipid peroxidation of LDL and the measurement of free radical scavenging capacity using stable free radical, 1,1,-diphenyl-2-picrylhydrazyl, demonstrated that the o-dihydroxyl structure in the B ring is required to exert maximum free radical scavenging activity. It is therefore likely that conjugation occurs at least partly in positions other than the B ring during the process of metabolic conversion so that the inhibitory effect of dietary quercetin is retained in blood plasma after absorption.

Phloretin-induced apoptosis in B16 melanoma 4A5 cells and HL60 human leukemia cells.

The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells.

Proanthocyanidins from barley bran potentiate retinoic acid-induced granulocytic and sodium butyrate-induced monocytic differentiation of HL60 cells.

Polyphenol extract from barley bran (BPE) induced nitro blue tetrazolium (NBT) reducing activity and alpha-naphthyl butyrate esterase activity in HL60 human myeloid leukemia cells. Because BPE induced the biochemical markers of HL60 cell differentiation, we investigated the effects of proanthocyanidins isolated from BPE on the HL60 cell differentiation of HL60 cells. Prodelphinidin B-3, T1, T2, and T3 induced 26-40% NBT-positive cells and 22-32% alpha-naphthyl butyrate esterase-positive cells. Proanthocyanidins potentiated retinoic acid (all-trans-retinoic acid)-induced granulocytic and sodium butyrate-induced monocytic differentiation in HL60 cells.

Inhibition of mammalian 15-lipoxygenase-dependent lipid peroxidation in low-density lipoprotein by quercetin and quercetin monoglucosides.

Lipoxygenase is suggested to be involved in the early event of atherosclerosis by inducing plasma low-density lipoprotein (LDL) oxidation in the subendothelial space of the arterial wall. Since flavonoids such as quercetin are recognized as lipoxygenase inhibitors and they occur mainly in the glycoside form, we assessed the effect of quercetin and its glycosides (quercetin 3-O-beta-glucopyranoside, Q3G; quercetin 4'-O-beta-glucopyranoside, Q4'G; quercetin 7-O-beta-glucopyranoside, Q7G) on rabbit reticulocyte 15-lipoxygenase (15-LOX)-induced human LDL lipid peroxidation and compared it with the inhibition obtained by ascorbic acid and alpha-tocopherol, the main water-soluble and lipid-soluble antioxidants in blood plasma, respectively. Quercetin inhibited the formation of cholesteryl ester hydroperoxides (CE-OOH) and endogenous alpha-tocopherol consumption effectively throughout the incubation period of 6 h. Ascorbic acid exhibited an effective inhibition only in the initial stage and LDL preloaded with fivefold alpha-tocopherol did not affect the formation of CE-OOH compared with the native LDL. CE-OOH formation was inhibited by both quercetin and quercetin monoglucosides in a concentration-dependent manner. Quercetin, Q3G, and Q7G exhibited a higher inhibitory effect than Q4'G (IC50: 0.3-0.5 microM for quercetin, Q3G, and Q7G and 1.2 microM for Q4'G). While endogenous alpha-tocopherol was completely depleted after 2 h of LDL oxidation, quercetin, Q7G, and Q3G prevented the consumption of alpha-tocopherol. Quercetin and its monoglucosides were also exhausted during the LDL oxidation. These results indicate that quercetin glycosides as well as its aglycone are capable of inhibiting lipoxygenase-induced LDL oxidation more efficiently than ascorbic acid and alpha-tocopherol.

Structure-activity relationships of flavonoids and the induction of granulocytic- or monocytic-differentiation in HL60 human myeloid leukemia cells.

The flavones apigenin and luteolin strongly inhibited the growth of HL60 cells and induced morphological differentiation into granulocytes. The flavonol quercetin inhibited the cell growth and induced a differentiation marker, i.e., NBT reducing ability. However quercetin-treated cells were not morphologically differentiated into granulocytes. The chalcone phloretin weakly induced NBT reducing ability and a marker of monocytic differentiation alpha-naphthyl butyrate esterase activity in the cells. Quercetin and phloretin appeared to induce the differentiation of HL60 cells into monocytes. The proportion of alpha-naphthyl butyrate esterase-positive cells induced by genistein was less than that of the NBT-positive cells. Some of the nuclei in genistein-treated HL60 cells morphologically changed. Genistein must have induced both granulocytic and monocytic differentiation of HL60 cells. The flavonols galangin and kaempferol, which had fewer hydroxyl group(s) in the B-ring than quercetin, and the flavanone naringenin inhibited the growth but did not induce the differentiation of HL60 cells.

Stimulation of cell growth in the U-937 human myeloid cell line by honey royal jelly protein.

Royal jelly was fractionated by ion-exchange chromatography and a protein (DIII protein) that had growth stimulating activity to the U-937 human myeloid cell line was obtained. The molecular weight of the DIII protein was 58 kDa on SDS-PAGE. The growth stimulating activity of the DIII protein was shown to be relatively heat and pH stable.

Competitive ELISA of bovine lactoferrin with bispecific monoclonal antibodies.

A mouse hybrid-hybridoma, HH1-4-3, secreting IgG1 class bispecific antibodies to bovine lactoferrin (bLF) and horseradish peroxidase (HRPO), was established previously. The competitive enzyme-linked immunosorbent assay (ELISA) of bLF using the HH1-4-3 culture supernatant was not sensitive enough to measure bLF concentration in biological fluids. To improve the sensitivity of the competitive ELISA, we fractionated the bispecific antibodies by antigen affinity column chromatography. A column immobilized with bLF adsorbed 60% of the antibodies of the HH1-4-3 supernatant, and the amount of antibodies adsorbed on a column immobilized with HRPO was less than 5%. The competitive ELISA of bLF using the affinity purified bispecific antibodies through an HRPO-immobilized column chromatography showed a good standard curve at bLF concentrations of 10 ng/ml to 100 micrograms/ml.

Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport.

Phloretin, a naturally occurring dihydrochalcone, is known to inhibit tumor cell growth in vitro and in vivo. To clarify the anti-tumor effects of phloretin, its apoptosis-inducing effects in B16 melanoma 4A5 cells were examined. Phloretin induced the internucleosomal DNA fragmentation typical of apoptosis in B16 melanoma cells. The addition of extracellular glucose remarkably inhibited the phloretin-induced apoptosis in the cells. When apoptosis was strongly induced in the B16 cells by phloretin, protein kinase C activity was inhibited in the cells. Our results suggest that phloretin induced apoptosis in B16 melanoma 4A5 cells mainly through the inhibition of glucose transmembrane transport. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in the cells.

Progressive effects of phloridzin on melanogenesis in B16 mouse melanoma cells.

When we studied the effects of polyphenols from apple fruits on melanogenesis in B16 mouse melanoma cell lines, phloridzin had dose-dependent progressive effects on melanogenesis between 10 and 500 micrograms/ml without inhibiting cell growth. At a concentration of 500 micrograms/ml, phloridzin increased the melanin content in the cells to 181% of that in control cells. In contrast, phloretin, the aglycon of phloridzin, did not activate melanogenesis in the cells and was cytotoxic at a concentration of 5 micrograms/ml. Phloridzin increased the activity of tyrosinase to 223% of that in control cells. Furthermore, phloridzin inhibited the activity of protein kinase C (PKC), which is recognized to regulate tyrosinase activity. The inhibition of PKC activity continued for 120 min from the addition of phloridzin. Therefore, we estimated that the activation of melanogenesis by phloridzin resulted from the increase of tyrosinase activity caused by the inhibition of PKC activity.

Assessment of the mutagenicity of extracts of TMV-coat-protein-gene induced transgenic tomato by the umu-test.

We examined the mutagenicity of extracts (juice and ethanol extract) from a transgenic tomato that was established by transfection of a gene encoding the coat protein of tobacco mosaic virus (TMV) to the F1 hybrid between Lycopersicon esculentum LA1000 and L. peruvianum PI128650, by the umu-test with Salmonella typhimurium TA1535/pSK1002 as the test organism. The extracts showed no detectable mutagenicity. The extracts from the above-mentioned F1 hybrids and wild tomatoes and cultivars (L. peruvianum PI128650, L. peruvianum PI126944, L. pimpinellifolium LS1524, L. pimpinellifolium LA722, L. hirsutum LS503, Mini-carol, Sun-cherry, Momotaro, Odoriko, Kagome77, and Ponderosa) also showed no detectable mutagenicity.

Antioxidative activity of quercetin and quercetin monoglucosides in solution and phospholipid bilayers.

The antioxidative effect of quercetin, quercetin 3-O-beta-D-glucopyranoside (Q3G), quercetin 4'-O-beta-D-glucopyranoside (Q4'G) and quercetin 7-O-beta-D-glucopyranoside (Q7G) was examined in solution and liposomal phospholipid suspension. First, their peroxyl radical-scavenging activities were investigated by measuring the inhibition of hydroperoxidation of methyl linoleate initiated by a radical initiator, 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Quercetin exhibited the highest peroxyl radical-scavenging activity judging from the rate of hydroperoxidation during the induction period (Rinh) and the length of induction period (tinh). Although Q7G showed an induction period, its Rinh was higher and its tinh was lower than that of quercetin. Neither Q3G nor Q4'G gave a clear induction period in the curve of hydroperoxide formation. The rate of hydroperoxidation in the presence of Q3G was higher than Rinh of quercetin and the oxidative loss of Q3G was much slower than quercetin or Q7G when exposed to AMVN in solution. Q4'G exerted little inhibition compared to Q3G or Q7G. Next, the antioxidative activity of quercetin and its monoglucosides in phospholipid bilayers was examined by measuring the inhibition of lipid peroxidation in large unilamellar vesicles composed of egg yolk phosphatidylcholine (PC) and a water-soluble radical initiator. They retarded the accumulation of PC-hydroperoxides and the induction period increased in the order of Q4'G < Q3G approximately Q7G < quercetin. It is therefore concluded that quercetin acts as an antioxidant more efficiently than its monoglucosides when phospholipid bilayers are exposed to aqueous oxygen radicals.